Orotidylate decarboxylase: insights into the catalytic mechanism from substrate specificity studies

Shostak, Keith; Jones, Mary Ellen

doi:10.1021/bi00163a026

Cited by 49 publications

(75 citation statements)

References 30 publications

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“…Replacement of the C4 carbonyl group of the substrate with a thiocarbonyl group (4-thioOMP) has only a small effect on k cat (50% reduction). In contrast, the C2 thiocarbonyl containing analog (2-thioOMP) is not a substrate (k cat is reduced by at least 5,000-fold) (8). This suggests that hydrogen bonding to the C2 carbonyl is much more important in stabilizing the transition state for the decarboxylation than hydrogen bonding to the C4 carbonyl.…”

Section: Resultsmentioning

confidence: 99%

“…This intriguing mechanism was suggested based on theoretical calculations (5-7). However, this mechanism does not explain why the replacement of the C4 carbonyl with a thiocarbonyl has only a small effect on the reaction rate (50% reduction in k cat ) whereas replacement of the C2 carbonyl group with a thiocarbonyl has a large effect on the rate (k cat reduced by Ͼ10,000-fold) (8).…”

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confidence: 96%

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The crystal structure and mechanism of orotidine 5′-monophosphate decarboxylase

Appleby

Kinsland

Begley

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

172

216

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The crystal structure of Bacillus subtilis orotidine 5-monophosphate (OMP) decarboxylase with bound uridine 5-monophosphate has been determined by multiple wavelength anomalous diffraction phasing techniques and refined to an R-factor of 19.3% at 2.4 Å resolution. OMP decarboxylase is a dimer of two identical subunits. Each monomer consists of a triosephosphate isomerase barrel and contains an active site that is located across one end of the barrel and near the dimer interface. For each active site, most of the residues are contributed by one monomer with a few residues contributed from the adjacent monomer. The most highly conserved residues are located in the active site and suggest a novel catalytic mechanism for decarboxylation that is different from any previously proposed OMP decarboxylase mechanism. The uridine 5-monophosphate molecule is bound to the active site such that the phosphate group is most exposed and the C5-C6 edge of the pyrimidine base is most buried. In the proposed catalytic mechanism, the ground state of the substrate is destabilized by electrostatic repulsion between the carboxylate of the substrate and the carboxylate of Asp60. This repulsion is reduced in the transition state by shifting negative charge from the carboxylate to C6 of the pyrimidine, which is close to the protonated amine of Lys62. We propose that the decarboxylation of OMP proceeds by an electrophilic substitution mechanism in which decarboxylation and carbon-carbon bond protonation by Lys62 occur in a concerted reaction. O rotidine monophosphate (OMP, 1) decarboxylase catalyzes the final step in the de novo biosynthesis of uridine monophosphate (UMP, 2) (Eq. 1).In most prokaryotes, OMP decarboxylase is a dimer of identical subunits whereas in higher organisms, it is part of a bifunctional enzyme that also catalyzes the formation of OMP. Amino acid sequence comparisons suggest that monomeric and bifunctional OMP decarboxylases are structurally homologous with about a dozen residues conserved throughout all species. The enzyme accelerates this decarboxylation reaction by 10 17 and is the most proficient enzyme identified so far (1). OMP decarboxylase does not use any cofactors (2). Its mechanism is novel because the carbanion generated by carbon dioxide loss is localized in an sp 2 orbital perpendicular to the system of the pyrimidine. In all other decarboxylases, the carbanion is delocalized either into an adjacent carbonyl or into a covalently bound thiamin, pyridoxal, or pyruvoyl cofactor (3). Although several hypotheses have been advanced to explain how the enzyme stabilizes the carbanion intermediate, the mechanistic details of this reaction are currently unclear.Three mechanisms have been proposed for OMP decarboxylase (Scheme 1). In the first mechanism (zwitterion mechanism), protonation of the C2 carbonyl group would generate the zwitterion 3, in which the positive charge at N1 could stabilize the negative charge accumulating at C6 during the decarboxylation. This proposal was supported by a model study that dem...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 96%

The crystal structure and mechanism of orotidine 5′-monophosphate decarboxylase

Appleby

Kinsland

Begley

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

172

216

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“…15,44 Reactions were performed at 25 C in MOPS buffer, pH ¼ 7.2 in a total volume of 0.3 mL and monitored in a Beckman DU 800 spectrophotometer. The wild-type ODCase concentration used was 3.9 nM.…”

Section: Determination Of Kinetic Parametersmentioning

confidence: 99%

Determination of the amino acid sequence requirements for catalysis by the highly proficient orotidine monophosphate decarboxylase

Yuan¹,

Cárdenas²,

Gilbert³

et al. 2011

Protein Science

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Orotidine 5 0 -monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine 5 0 -monophosphate to uridine 5 0 -monophosphate during pyrimidine nucleotide biosynthesis. This enzyme is one of the most proficient known, exhibiting a rate enhancement of over 17 orders of magnitude over the uncatalyzed rate. An interesting question is whether the high proficiency of ODCase is associated with a highly optimized sequence of active site residues. This question was addressed by randomizing 24 residue positions in and around the active site of the E. coli ODCase (pyrF) by site-directed mutagenesis. The libraries of mutants were selected for function from a multicopy plasmid or by single-copy replacement at the pyrF locus on the E. coli chromosome. Stringent sequence requirements for function were found for the mutants expressed from the chromosomal pyrF locus. Six positions were not tolerant of substitutions and several others accepted very limited substitutions. In contrast, all positions could be substituted to some extent when the library mutants were expressed from a multicopy plasmid. For the conserved quartet of charged residues Lys44-Asp71-Lys73-Asp76, a cysteine substitution was found to provide function at positions 71 and 76. A lower pK a for both cysteine mutants supports a mechanism whereby the thiolate group of cysteine substitutes for the negatively charged aspartate side chain. The partial function mutants such as D71C and D76C exhibit reduced catalytic efficiency relative to wild type but nevertheless provide a rate enhancement of 15 orders of magnitude over the uncatalyzed rate indicating the catalytic proficiency of the enzyme is robust and tolerant of mutation.

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“…14 Michael addition of an active site nucleophilic residue at C5 was suggested early on by Silverman and Groziak. 15 The importance of an active site lysine led Shostack and Jones to propose Schiff base formation at C4, 16 but lack of 18 O exchange of O4 with water seemed to rule that out.…”

Section: Introductionmentioning

confidence: 99%

QM/MM Metadynamics Study of the Direct Decarboxylation Mechanism for Orotidine-5‘-monophosphate Decarboxylase Using Two Different QM Regions: Acceleration Too Small To Explain Rate of Enzyme Catalysis

et al. 2007

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Despite decades of study, the mechanism by which orotidine-5'-monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine monophosphate remains unresolved. A computational investigation of the direct decarboxylation mechanism has been performed using mixed quantum mechanical/molecular mechanical (QM/MM) dynamics simulations. The study was performed with the program CP2K that integrates classical dynamics and ab initio dynamics based on the Born-Oppenheimer approach. Two different QM regions were explored. The free energy barriers for decarboxylation of orotidine-5'-monophosphate (OMP) in solution and in the enzyme (using the larger QM region) were determined with the metadynamics method to be 40 kcal/mol and 33 kcal/mol, respectively. The calculated change in activation free energy (ΔΔG ± ) on going from solution to the enzyme is therefore −7 kcal/mol, far less than the experimental change of −23 kcal/mol (for k cat /k uncat Radzicka, A.; Wolfenden, R., Science. 1995, 267, 90-92). These results do not support the direct decarboxylation mechanism that has been proposed for the enzyme. However, in the context of QM/MM calculations, it was found that the size of the QM region has a dramatic effect on the calculated reaction barrier.

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Orotidylate decarboxylase: insights into the catalytic mechanism from substrate specificity studies

Cited by 49 publications

References 30 publications

The crystal structure and mechanism of orotidine 5′-monophosphate decarboxylase

The crystal structure and mechanism of orotidine 5′-monophosphate decarboxylase

Determination of the amino acid sequence requirements for catalysis by the highly proficient orotidine monophosphate decarboxylase

QM/MM Metadynamics Study of the Direct Decarboxylation Mechanism for Orotidine-5‘-monophosphate Decarboxylase Using Two Different QM Regions: Acceleration Too Small To Explain Rate of Enzyme Catalysis

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